Document Type

Thesis - Open Access

Award Date

2023

Degree Name

Master of Science (MS)

Department / School

Dairy and Food Science

First Advisor

Sanjeev Anand

Keywords

Biofilm, Brick floors, Dairy plant, Listeria monocytogenes, Persistence, Whole Genome Sequencing

Abstract

Listeria monocytogenes is a prevalent foodborne pathogen that forms biofilms on floors in dairy processing facilities and can potentially cross-contaminate processed products. Despite the fact that these facilities have comprehensive floor cleaning systems, L. monocytogenes can still thrive. The primary objective of the first study was to assess the efficacy of a typical floor cleaning protocol commonly employed in a commercial dairy plant for eliminating biofilms of L. monocytogenes that had developed on brick flooring. In addition, the study also evaluated brick floors that were both new and 15 years old to investigate the impact of surface alterations over time on the formation of L. monocytogenes biofilms. The study reports that the surface roughness (Ra) values for new and old brick floors were 4.99 ± 0.39 μm and 5.82 ± 0.49 μm, respectively. Even though a difference in Ra values was reported among the new and old floor surfaces, it did not appear to influence L. monocytogenes biofilm formation on the respective floors. Further, in a simulated dairy plant setting, L. monocytogenes exhibited the greatest tendency for biofilm formation at a temperature of 22 °C, with lower levels of biofilm formation observed at temperatures of 37 °C, followed by 7 °C. The cleanability of L. monocytogenes biofilms formed at temperatures of 37 °C, 22 °C, and 7 °C on both new and old floors was evaluated at 24 h, 48 h, and 72 h intervals. The findings indicated that the implementation of cleaning and sanitizing protocols led to a consistent 3-log reduction in biofilm counts at 22 °C and 7 °C across all time intervals. However, survivor cells were still observed following cleaning procedures conducted at 7 °C after 48 h and 72 h, as well as at 22 °C after 24 h, 48 h, and 72 h. Nevertheless, despite the sanitizer currently being utilized exhibiting a notable sensitivity towards the planktonic counterparts, it was unable to achieve a reduction of 3-logs at a temperature of 37 °C for all durations. Attaining a reduction of three logarithmic units or more aligns with the guidelines established by the Environmental Protection Agency (EPA). The higher exopolymeric substance (EPS) formation at 37 °C, as reported by previous researchers, could account for the lack of similar observations regarding L. monocytogenes biofilm cleanability to that of 22 °C and 7 °C. Higher EPS formation is likely to cause an increase in biofilm layer thickness and potentially impede the cleaning protocol. The findings of this investigation demonstrate that the incubation time and temperature are significant factors for biofilm formation, rather than surface roughness. Based on the above information, it is apparent that some cells of L. monocytogenes may survive despite undergoing a 3-log reduction at temperatures of 22 °C and 7 °C, after cleaning procedures conducted at intervals of 48 hours, and 7 hours, and 24 hours, 48 hours, and 72 hours on biofilms, respectively. Indicating the persistent nature of the pathogen, despite the implementation of rigorous floor cleaning protocols. However, the molecular mechanisms driving this pathogen's persistence are currently unknown. Therefore, to gain an improved understanding of the persistence mechanism of L. monocytogenes, it is imperative to produce accurate and continuous genome assemblies, which will enable the acquisition of reliable downstream analyses, such as gene annotations. The second investigation involved the sequencing of 10 distinct Listeria isolates, utilizing the Illumina MiSeq and Oxford Nanopore MinION sequencing technologies. The primary objective was to evaluate the individual and combined benefits of each approach, with the latter involving the correction of Nanopore assemblies with Illumina short reads. Despite the high accuracy of the assemblies produced by Illumina reads, they exhibited a deficiency in contiguity. Enhanced genome contiguity has the potential to significantly enhance the annotation of persistence genes in Listeria species. This is due to the ability of long reads to provide insight into genomic regions that are highly repetitive yet crucial. The assemblies that exclusively utilized MinION reads exhibited a high level of continuity, albeit with a notable lack of accuracy. However, this limitation was partially mitigated by correcting the long read assemblies with Illumina short reads. The corrected assembly exhibited enhanced assembly properties and facilitated the detection of multiple new alleles among the Listeria isolates. The second objective of this study was to compare the genetic components found through the annotation of persistence genes in the corrected assembly in both inter- and intra-isolate variations among Listeria species. The study revealed that the L. monocytogenes of dairy plant origin (L. monocytogenes 315-S-1) exhibited the highest count of persistent genes in comparison to other Listeria species, specifically in relation to biofilm-forming genes. L. monocytogenes 315-S-1 possessed a total of 12 biofilm-forming genes, whereas the other species and strains only contained between 0-1 genes. L. monocytogenes has been reported to have a high degree of receptivity towards mobile genetic elements, thereby enhancing its ability to endure and persist even under adverse environmental conditions. The results of this study enhance our comprehension of the persistent nature of L. monocytogenes in dairy facilities.

Library of Congress Subject Headings

Listeria monocytogenes -- Control.
Pathogenic bacteria -- Control.
Dairy products -- Contamination.
Dairy plants -- Sanitation.
Floors.
Genomics.
Phenotype.

Publisher

South Dakota State University

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